This application is phase I of a FAST TRACK SBIR grant proposal. The primary goal of this study is to generate a novel glioma cell vaccine comprised of six well-characterized allogeneic glioma cell lines in whichTGF-beta1 and TGF-beta2 secretion has been abrogated using gene knockout technology. The vaccine generated in this manner will be used to immunize patients with stage IV astrocytoma (glioblastoma multiforme and gliosarcoma) in a Phase II/III clinical trial with the goal of inducing antitumor immunity that leads to clinical responses, to be partially funded by the accompanying phase II grant proposal. By blocking secretion of the immunosuppressive TGF-beta molecules in this manner, we inhibit one of the major mechanisms by which tumor cells evade immune surveillance. Development of an effective therapy for this disease will benefit the approximately 20,000 new patients that develop glial tumors in the United States each year. The milestone for progressing to the phase II SBIR proposal will be successful generation and certification of master and working vaccine cell banks. The first aim of the study is to completely abrogate expression TGF-beta1 andTGF-beta2 by knocking out the paternal and maternal copies for TGF-beta1 and TGF-beta2 genes in six allogeneic glioma cell lines. Candidate cells chosen from eighteen malignant glioma cell lines carried in the cryopreservation inventory of NovaRx will be transfected with the constructed TGF-beta1 and TGF-beta2 knockout vectors. Following selection of cells in the presence of designated drugs, the surviving cell colonies will be expanded and characterized to ascertain the correct incorporation of the recombinant unit and expression of glioma-associated antigens. The lack of expression of TGF-beta1 and TGF-beta2 will be confirmed by Northern and Southern blot analyses, ELISA, and Western blot analyses. Additionally, we will characterize the gene-modified cells by HLA and glial cell marker phenotyping. We are expecting of a stable 100% down-regulation of TGF-beta1 and TGF-beta2 secretion. The second aim of the study is to generate master and working cell banks of the six allogeneic cell lines generated in Aim 1. The cell banks will be tested for the presence of bacteria, fungi, adventitious viruses, mycoplasma, and endotoxin in order to certify them for use in a Phase II/III clinical trial. Completion of Aims l and 2 serves as a milestone for initiation of the Phase II study. We anticipate that genetic modification of malignant glioma cells to block TGF-beta expression would result in the gene-modified cells becoming more immunogenic and suitable for active tumor immunotherapy.